Method for counterselection in microorganisms

ABSTRACT

The present disclosure is directed to methods of scarless genomic engineering in microorganisms, such as  Bacillus , and provides for new molecular tools and methods which enable scarless genetic editing using at least one counterselectable marker that has been codon optimized for the microorganism. The disclosure allows for the high-throughput introduction of stable genetic edits to a genome using either plasmid or linear DNA constructs for genetic engineering.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/904,285 filed on Sep. 23, 2019, which is herebyincorporated by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is ZYMR_048_01WO_SeqList_ST25. The text file of 4kb was created on Sep. 21, 2020, and is being submitted electronicallyvia EFS-Web.

TECHNICAL FIELD

The present disclosure is directed to methods of scarless genomicengineering in microorganisms, such as Bacillus, and provides for newmolecular tools and methods which enable scarless genetic editing usingat least one counterselectable marker that has been codon optimized forthe microorganism. The disclosure allows for the high-throughputintroduction of stable genetic edits to a genome using either plasmid orlinear DNA constructs for genetic engineering.

BACKGROUND

Previous work in E. coli has demonstrated that two mutations(A294G/T251A or A294G/T251S) to the sequence the α-subunit ofphenylalanyl-tRNA ligase, PheS, can be used as a counterselectablemarker due to the incorporation of 4-chloro-DL-phenylalanine (4CP) inplace of phenylalanine during translation, leading to cell death (Kast,Hennecke, 1991, Amino acid substrate specificity of Escherichia coliphenylalanyl-tRNA synthetase altered by distinct mutations, J Mol Biol.222(1):99-124, Miyazaki, 2105, Molecular engineering of a PheScounterselection marker for improved operating efficiency in Escherichiacoli, Biotechniques, 58(2):86-8). However, in Bacillus species thosesame PheS mutations are not effective.

When PheS sequences were aligned and the corresponding mutations,A309G/T255S, were identified and introduced into B. subtilis pheS, andexpressed in B. amyloliquefaciens heterologously, this resulted inefficient killing in the presence of 4CP (Kharchenko M. S. et al., 2018.Improving the selection efficiency of the counter-selection marker pheS*for the genetic engineering of Bacillus amyloliquefaciens, J MicrobiolMethods, 148:18-21, and Zhou, C. et al., 2017, pheS*, an effectivehost-genotype-independent counter-selectable marker for marker-freechromosome deletion in Bacillus amyloliquefaciens. Appl MicrobiolBiotechnol, 101(1): 217-227). However, expression of the mutant gene inB. subtilis resulted in false-positive clones due to homologousrecombination that replaced the mutant gene with the chromosomalwild-type pheS.

In the absence of an effective counterselectable marker, loop-out ratesfor Bacillus range from 0.8% to 5%, making the reliable construction ofgenomic edits very challenging, especially in a high-throughput (HTP)context. Although some effective counterselection markers have beenreported in the literature for Bacillus species, many of those requirepre-existing genetic mutations such as upp, which requires the nativeupp be deleted from the genome for counterselection with 5-fluorouracil(Dong and Zhang, 2014, Current development in genetic engineeringstrategies of Bacillus species, Microb Cell Fact. 13:63). Similarly, useof pyrF as a counterselection marker, requires that both pyrF and pyrRbe deleted in the strain of interest. Thus, there is a need in the artfor new molecular tools adapted to enable greater precision, efficiency,and predictability when editing the genome of Bacillus.

SUMMARY

In one embodiment, the disclosure teaches a high-throughput (HTP) methodfor generating at least one scarless genomic edit in a microorganism,comprising: providing a plasmid or linear DNA construct comprising asequence of interest, a means for positive selection, and twocounterselectable markers, wherein each of the counterselectable markershave been independently codon optimized for the microorganism and have amaximum tandem identity length of 500 base pairs when aligned with eachother, and wherein each counterselectable marker is operably linked toat least one promoter; transforming the microorganism with the DNAconstruct; selecting for a microorganism strain having integrated theDNA construct based on the means for positive selection; selecting for amicroorganism having undergone a homologous recombination event excisingthe backbone of the plasmid containing the counterselectable markers toproduce a loop-out strain; screening the loop-out strain for thepresence of the sequence of interest to produce a modified microorganismhaving at least one scarless genomic edit.

In another embodiment, the disclosure teaches a HTP method forgenerating at least one scarless genomic edit in a microorganismcomprising providing a plasmid or linear DNA construct comprising asequence of interest and at least one counterselectable marker, whereinthe at least one counterselectable marker is a homolog of the α-subunitof Phenylalanyl-tRNA ligase, (PheS) that has been codon optimized for amicroorganism, and further comprises homologous mutations correspondingto A309G/T255S of Bacillus PheS, and wherein the at least onecounterselectable marker is operably linked to at least one promoter;transforming the microorganism with the DNA construct to produce atransformed strain; growing the transformed strain in the presence of4-chlorphenylalanine to select for a strain having undergone arecombination event excising the backbone of the plasmid containing theat least one counterselectable marker to produce a loop-out strain;screening the loop-out strain for the presence of the sequence ofinterest to produce a scarless genetically modified microorganism. Inanother embodiment, the DNA construct comprises two counterselectablemarkers, wherein the markers have been independently codon optimized forthe microorganism and are sufficiently distinct to prevent homologousrecombination between the two markers.

In another embodiment, the present disclosure teaches a HTP method forgenerating at least one scarless genomic edit in a Bacillus speciescomprising providing a plasmid or linear DNA construct comprising asequence of interest, a means for positive selection, and acounterselectable marker, wherein the counterselectable marker is anα-subunit of phenylalanyl-tRNA ligase, (PheS) that has been codonoptimized for Bacillus and further comprises A309G/T255S mutations, andwherein the counterselectable marker is operably linked to at least onepromoter; transforming a Bacillus species with the DNA construct;selecting a Bacillus strain having integrated the DNA construct based onthe means for positive selection; growing the Bacillus strain havingintegrated the DNA construct in the presence of 4-chlorphenylalanine toselect for a Bacillus strain having undergone a homologous recombinationevent excising the backbone of the plasmid containing thecounterselectable marker to produce a loop-out strain; and screening theloop-out strain for the presence of the sequence of interest to producea modified Bacillus strain having at least one scarless genomic edit.

In another embodiment, the disclosure provides for a method forgenerating at least one scarless genomic edit in a Bacillus species,comprising: providing a plasmid or linear DNA construct comprising asequence of interest, a means for positive selection, and twocounterselectable markers, wherein the counterselectable markers are anα-subunit of phenylalanyl-tRNA ligase, (PheS) that have been codonoptimized for Bacillus and further comprise A309G/T255S mutations,wherein the counterselectable markers have a maximum tandem identitylength of 500 base pairs when aligned with each other, and wherein eachcounterselectable marker is operably linked to at least one promoter;transforming a Bacillus species with the DNA construct; selecting for aBacillus strain having integrated the DNA construct based on the meansfor positive selection; growing the Bacillus strain having integratedthe DNA construct in the presence of 4-chlorphenylalanine to select fora Bacillus strain having undergone a homologous recombination eventexcising the backbone of the plasmid containing the counterselectablemarker to produce a loop-out strain; and screening the loop-out strainfor the presence of the sequence of interest to produce a modifiedBacillus strain having at least one scarless genomic edit.

In some embodiments, the PheS that has been codon optimized for Bacillusis selected from the group consisting of SEQ ID NO: 1 (herein afterreferred to as PheS(**CO)), or a sequence at least 90% identicalthereto, and SEQ ID NO: 2 (herein after referred to as PheS(**), or asequence at least 75% identical thereto.

In some embodiments, the at least one promoter operably linked to thecounterselectable marker is constitutive, inducible, differentiallyinducible, endogenous, heterologous, synthetic, a dual promoter, or atandem promoter cluster. In some embodiments, the promoter is selectedfrom the group consisting of PliaG, P43, PliaI, PrpsF, Pspac, andPspank.

In some embodiments, the DNA construct further comprises a terminationsequence. In some embodiments, the termination sequence is selected fromthe group consisting of TgyrA, Tsero_aroC, and TcodBA.

While it is contemplated that the methods can be applied to and/orutilized in any Bacillus species, in one embodiment, the Bacillusspecies is selected from the group consisting of B. coagulans, B.ginsengihumi, B. shackletonii, B. aerius, B. aerophilus, B.stratosphericus, B. licheniformis, B. sonorensis, B. amyloliquefaciens,B. velezensis, B. atrophaeus, B. pumilus, B. safensis, B. altitudinis,B. vallismortis, B. subtilis, B. tequilensis, B. mojavensis, B.carboniphilus, B. oleronius, B. sporothermodurans, B. acidicola, B.aquimaris, B. vietnamensis, B. marisflavi, B. seohaeanensis, B.endophyticus, and B. humi

While it is contemplated that the strain having integrated the DNAconstruct can be grown on or in media containing 4-chlorphenylalanine,in one embodiment the strain is grown on or in media containing between1 mM and 20 mM 4-chlorphenylalanine.

While it is contemplated that a number of screening methods can be usedwith the methods disclosed herein, in one embodiment the screening ofthe loop-out strain comprises sequencing, DNA fingerprinting, orphenotypic analysis.

In some embodiments, the sequence of interest is an endogenous gene,wherein the endogenous gene has at least one mutation sequence, or aheterologous gene. In some embodiments, the mutation sequence comprisesa mutation selected from the group consisting of: a single nucleotideinsertion, an insertion of two or more nucleotides, an insertion of anucleic acid sequence encoding one or more proteins, a single nucleotidedeletion, a deletion of two or more nucleotides, a deletion of one ormore coding sequences, a substitution of a single nucleotide, asubstitution of two or more nucleotides, two or more non-contiguousinsertions, deletions, and/or substitutions, and any combinationthereof.

In some embodiments, the disclosure provides for a genetically modifiedBacillus strain produced by the methods disclosed herein. In someembodiments, the modified Bacillus strain produced is subjected tofurther genetic modification.

In some embodiments, the present disclosure provides for a DNA constructcomprising at least one of a counterselectable marker comprising SEQ IDNO: 1, or a sequence at least 90% identical thereto, and acounterselectable marker comprising SEQ ID NO: 2, or a sequence at least75% identical thereto.

In some embodiments, the present disclosure provides for an isolatednucleic acid comprising SEQ ID NO: 1, or a sequence at least 90%identical thereto, or SEQ ID NO: 2, or a sequence at least 75% identicalthereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures, which are incorporated herein and form a partof the specification, illustrate some, but not the only or exclusive,example embodiments and/or features. It is intended that the embodimentsand figures disclosed herein are to be considered illustrative ratherthan limiting.

FIG. 1A-FIG. 1D show photographs of a culture dilution series of B.subtilis strain S30A transformed with plasmids bearing PheS(**CO) drivenby different promoters. Cultures were plated on lysogeny broth (LB)alone (FIG. 1A), selective antibiotic (chloramphenicol [Chlor]) (FIG.1B), counterselection reagent 4CP (FIG. 1C), or the combination of both(FIG. 1D).

FIG. 2A and FIG. 2B show photographs of a culture dilution series of B.subtilis strain 168 transformed with plasmids bearing PheS(**CO) drivenby different promoters. Cultures were plated on LB plates havingselective antibiotic (Erythromycin and Lincomycin [MLS]) (FIG. 2A), orMLS+ counterselection reagent 4CP (FIG. 2B).

FIG. 3A and FIG. 3B show photographs of a culture dilution series of B.licheniformis strain DSM13 transformed with plasmids bearing PheS(**CO)driven by different promoters. Cultures were plated on LB plates havingcounterselection reagent 4CP (FIG. 3A) or selective antibiotic MLS+4CP(FIG. 3B).

FIG. 4A and FIG. 4B show photographs of a culture dilution series of B.subtilis strain S30A without PheS(**CO) (FIG. 4A) or transformed withplasmids bearing PheS(**CO) driven by the PliaG promoter (FIG. 4B).Cultures were plated on media having counterselection reagent 4CP.

FIG. 5 is a nucleotide sequence alignment from EMBL-EBI, (EMBOSS Water,Smith-Waterman algorithm) between SEQ ID NO: 1 (PheS(**CO)) and SEQ IDNO: 2 (PheS(**)).

FIG. 6A and FIG. 6B show photographs of a culture dilution series of B.subtilis strain 530A transformed with one copy of PheS(**CO) driven byPliaG promoter and having a TgyrA termination sequence (FIG. 6A) and theother having two counterselectable markers, PheS(**CO) and PheS(**)driven by PliaG and TgyrA promoters, respectively, with TgryA andTserOaroC termination sequences, respectively (FIG. 6B). Cultures wereplated on media having counterselection reagent 4CP.

DEFINITIONS

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

The term “a” or “an” refers to one or more of that entity, i.e., canrefer to a plural referent. As such, the terms “a” or “an”, “one ormore” and “at least one” are used interchangeably herein. In addition,reference to “an element” by the indefinite article “a” or “an” does notexclude the possibility that more than one of the elements is present,unless the context clearly requires that there is one and only one ofthe elements.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device or themethod being employed to determine the value, or the variation thatexists among the samples being measured. Unless otherwise stated orotherwise evident from the context, the term “about” means within 10%(i.e., within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) above orbelow the reported numerical value (except where such number wouldexceed 100% of a possible value or go below 0%). When used inconjunction with a range or series of values, the term “about” appliesto the endpoints of the range or each of the values enumerated in theseries, unless otherwise indicated. As used in this application, theterms “about” and “approximately” are used as equivalents.

A “clone” is a population of cells derived from a single cell or commonancestor by mitosis. A “cell line” is a clone of a primary cell that iscapable of stable growth in vitro for many generations.

As used herein, the term “codon optimization” refers to a geneticengineering method wherein the codon bias of the host organism is usedto generate synonymous codon changes in a recombinant gene to increaseexpression and translation of the recombinant gene in the host.

The term “competent cell” refers to a cell which has the ability to takeup and replicate an exogenous nucleic acid.

As used herein, “counterselectable marker” or a “counterselectionmarker” is a nucleic acid segment that eliminates or inhibits growth ofa host organism upon selection. For example, it may render the cellssensitive to one or more chemicals/growth conditions/geneticbackgrounds.

As used herein, the term “endogenous” or “endogenous gene,” refers tothe natural sequence and/or location of a gene. In the context of thepresent disclosure, operably linking a heterologous promoter to anendogenous gene means genetically inserting a heterologous promotersequence in front of an existing gene, in the location where that geneis naturally present. An endogenous gene as described herein can includealleles of naturally occurring genes that have been mutated according toany of the methods of the present disclosure.

As used herein, the term “exogenous” is used interchangeably with theterm “heterologous,” and refers to a substance coming from some sourceother than its native source. For example, the terms “exogenousprotein,” or “exogenous gene” refer to a protein or gene from anon-native source or location, and that have been artificially suppliedto a biological system. Artificially mutated variants of endogenousgenes are considered “exogenous” for the purposes of this disclosure.

As used herein, an “extra-chromosomally replicating plasmid” is anautonomously replicating vector that exists as an extra-chromosomalentity. The replication of an extra-chromosomally replicating plasmid isindependent of chromosomal replication.

The term “genetic modification” or “mutation” refers to any alterationof DNA. Representative gene modifications include a “nucleotide change”such as insertions, deletions, substitutions, and combinations thereof,and can be as small as a single base or as large as tens of thousands ofbases. In some cases, mutations contain alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded protein or how the proteins are made. Theterm “genetic modification” also encompasses inversions of a nucleotidesequence and other chromosomal rearrangements, whereby the position ororientation of DNA comprising a region of a chromosome is altered. Achromosomal rearrangement can comprise an intrachromosomal rearrangementor an interchromosomal rearrangement.

As used herein, the term “heterologous” refers to an amino acid or anucleic acid sequence (e.g., gene or promoter), which is not naturallyfound in the particular organism or is not naturally found in aparticular context (e.g., genomic or plasmid location) in the particularorganism.

A “high-throughput (HTP)” method of genomic engineering may involve theutilization of at least one piece of automated equipment (e.g. a liquidhandler or plate handler machine) to carry out at least one step of themethod.

As used herein, the term “homologous” or “homologue” encompassesorthologs and paralogs and refers to related sequences that share acommon ancestor or family member and are determined based on the degreeof sequence identity. “Homologous sequences” or “homologs” may also befunctionally related. A functional relationship may be indicated in anyone of a number of ways, including, but not limited to: (a) degree ofsequence identity and/or (b) the same or similar biological function.Homology can be determined using software programs readily available inthe art, such as NCBI BLAST® (Basic Local Alignment Search Tool), usingdefault parameters, or using software programs readily available in theart, such as those discussed in Current Protocols in Molecular Biology(F. M. Ausubel et al., eds., 1987) Supplement 30, section 7.718, Table7.71. Some alignment programs are MacVector (Oxford Molecular Ltd,Oxford, U.K.), ALIGN Plus (Scientific and Educational Software,Pennsylvania) and AlignX (Vector NTI, Invitrogen, Carlsbad, Calif.).Another alignment program is Sequencher (Gene Codes, Ann Arbor, Mich.),using default parameters.

As used herein, the term “protein modification” refers to, e.g., aminoacid substitution, amino acid modification, deletion, and/or insertion,as is well understood in the art.

As used herein, the term “at least a portion” or “fragment” of a nucleicacid or polypeptide means a portion having the minimal sizecharacteristics of such sequences, or any larger fragment of the fulllength molecule, up to and including the full length molecule.

As used herein, the phrases “DNA construct”, “expression cassette”,“chimeric construct”, “construct”, and “recombinant DNA construct” areused interchangeably herein. A recombinant DNA construct comprises anartificial combination of nucleic acid fragments, e.g., regulatory andcoding sequences that are not found together in nature. For example, aconstruct may comprise regulatory sequences and coding sequences thatare derived from different sources, or regulatory sequences and codingsequences derived from the same source, but arranged in a mannerdifferent than that found in nature. Such construct may be used byitself or may be used in conjunction with a vector. If a vector is usedthen the choice of vector is dependent upon the method that will be usedto transform host cells as is well known to those skilled in the art.For example, a plasmid vector can be used.

The term “operably linked” refers to the juxtaposition of two or morecomponents (such as sequence elements) having a functional relationship.For example, the sequential arrangement of the promoter polynucleotidewith a further oligo- or polynucleotide, resulting in transcription ofthe further polynucleotide.

The term “product of interest” or “biomolecule” as used herein refers toany product produced by microbes. In some cases, the product of interestmay be a small molecule, enzyme, peptide, amino acid, organic acid,synthetic compound, fuel, alcohol, etc. For example, the product ofinterest or biomolecule may be any primary or secondary extracellularmetabolite.

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Insome embodiments, the promoter sequence consists of proximal and moredistal upstream elements, the latter elements often referred to asenhancers. Accordingly, an “enhancer” is a DNA sequence that canstimulate promoter activity, and may be an innate element of thepromoter or a heterologous element inserted to enhance the level ortissue specificity of a promoter.

As used herein, “selectable marker” is a nucleic acid segment thatallows one to select for a molecule (e.g., a plasmid) or a cell thatcontains it, often under particular conditions. These markers can encodean activity, such as, but not limited to, production of RNA, peptide, orprotein, or can provide a binding site for RNA, peptides, proteins,inorganic and organic compounds or compositions and the like.

A cell has been “transformed” or “transfected” when exogenous DNA hasbeen introduced inside the cell. The presence of the exogenous DNAresults in permanent or transient genetic change.

As used herein, the phrase “scarless” refers to a method of geneticengineering, also known as “scarless genomic editing” or “scarless genereplacement” wherein any markers (selectable and/or counterselectable)are removed, for example by recombination, from the transformedmicroorganism. Scarless may also be referred to as “clean” or “unmarked”mutations.

DETAILED DESCRIPTION

The following description includes information that may be useful inunderstanding the present disclosure. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed disclosures, or that any publication specifically orimplicitly referenced is prior art.

Overview

The disclosure provides for new molecular tools and methods, whichenable scarless genetic editing in a microorganism. The methods ofselection and counter selection, in combination with homologousrecombination events, are used to generate scarless mutations in amicroorganism in a two-step process. In the first step, a DNA cassette(or DNA construct) containing sequence of interest (SOI), a means forpositive selection, and counterselection marker (CSM) is introduced inthe host genome. The means for positive selection permits the selectionof cells that have incorporated the cassette. In a second step, theaction of the CSM exerts a negative pressure favoring a secondhomologous recombination event (loop-out) that results in a scarlessmutation.

An embodiment of the present disclosure teaches a HTP method forgenerating at least one scarless genomic edit in a microorganism,comprising: providing a plasmid or linear DNA construct comprising asequence of interest, a means for positive selection, and twocounterselectable markers, wherein each of the counterselectable markershave been independently codon optimized for the microorganism and have amaximum tandem identity length of 500 base pairs when aligned with eachother, and wherein each counterselectable marker is operably linked toat least one promoter; transforming the microorganism with the DNAconstruct; selecting for a microorganism strain having integrated theDNA construct based on the means for positive selection; selecting for amicroorganism having undergone a homologous recombination event excisingthe backbone of the plasmid containing the counterselectable markers toproduce a loop-out strain; screening the loop-out strain for thepresence of the sequence of interest to produce a modified microorganismhaving at least one scarless genomic edit.

In another embodiment, the disclosure teaches a HTP method forgenerating at least one scarless genomic edit in a microorganismcomprising providing a plasmid or linear DNA construct comprising asequence of interest and at least one counterselectable marker, whereinthe at least one counterselectable marker is a homolog of the α-subunitof Phenylalanyl-tRNA ligase, (PheS) that has been codon optimized for amicroorganism, and further comprises homologous mutations correspondingto A309G/T255S of Bacillus PheS, and wherein the at least onecounterselectable marker is operably linked to at least one promoter;transforming the microorganism with the DNA construct to produce atransformed strain; growing the transformed strain in the presence of4-chlorphenylalanine to select for a strain having undergone arecombination event excising the backbone of the plasmid containing theat least one counterselectable marker to produce a loop-out strain; andscreening the loop-out strain for the presence of the sequence ofinterest to produce a scarless genetically modified microorganism.

In addition to the PheS counterselectable marker described furtherbelow, other examples of such counterselectable marker genes includesacB, rpsL(strA), tetAR, pheS, thyA, gata-1, or ccdB, the function ofwhich is described in Reyrat et al. 1998 “Counterselectable Markers:Untapped Tools for Bacterial Genetics and Pathogenesis.” Infect Immun.,66(9): 4011-4017.

Microorganisms suitable for the methods disclosed herein include, butare not limited to, bacterial cells, algal cells, plant cells, fungalcells, insect cells, and mammalian cells. As used herein the terms“cellular organism” “microorganism” or “microbe” should be takenbroadly. These terms are used interchangeably and include, but are notlimited to, the two prokaryotic domains, Bacteria and Archaea, as wellas certain eukaryotic fungi and protists.

Codon Optimization

Protein expression is governed by a host of factors including those thataffect transcription, mRNA processing, and stability and initiation oftranslation. Optimization can thus address any of a number of sequencefeatures of any particular gene. Translation may be paused due to thepresence of codons in the polynucleotide of interest that are rarelyused in the host organism, and this may have a negative effect onprotein translation due to their scarcity in the available tRNA pool.Specifically, it can result in reduced protein expression.

Alternate translational initiation also can result in reducedheterologous protein expression. Alternate translational initiation caninclude a synthetic polynucleotide sequence inadvertently containingmotifs capable of functioning as a ribosome binding site (RBS). Thesesites can result in initiating translation of a truncated protein from agene-internal site. One method of reducing the possibility of producinga truncated protein includes eliminating putative internal RBS sequencesfrom an optimized polynucleotide sequence.

Repeat-induced polymerase slippage can result in reduced heterologousprotein expression. Repeat-induced polymerase slippage involvesnucleotide sequence repeats that have been shown to cause slippage orstuttering of DNA polymerase which can result in frameshift mutations.Such repeats can also cause slippage of RNA polymerase. In an organismwith a high G+C content bias, there can be a higher degree of repeatscomposed of G or C nucleotide repeats. Therefore, one method of reducingthe possibility of inducing RNA polymerase slippage, includes alteringextended repeats of G or C nucleotides.

Interfering secondary structures also can result in reduced heterologousprotein expression. Secondary structures can sequester the RBS sequenceor initiation codon and have been correlated to a reduction in proteinexpression. Stemloop structures can also be involved in transcriptionalpausing and attenuation. An optimized polynucleotide sequence cancontain minimal secondary structures in the RBS and gene coding regionsof the nucleotide sequence to allow for improved transcription andtranslation.

The optimization process can begin, for example, by identifying thedesired amino acid sequence to be expressed by the host. From the aminoacid sequence, a candidate polynucleotide or DNA sequence can bedesigned. During the design of the synthetic DNA sequence, the frequencyof codon usage can be compared to the codon usage of the host expressionorganism and rare host codons can be removed from the syntheticsequence. Additionally, the synthetic candidate DNA sequence can bemodified in order to remove undesirable enzyme restriction sites and addor remove any desired signal sequences, linkers or untranslated regions.The synthetic DNA sequence can be analyzed for the presence of secondarystructure that may interfere with the translation process, such as G/Crepeats and stem-loop structures.

Optimized coding sequences containing codons preferred by a particularprokaryotic or eukaryotic host (see also, Murray et al. (1989) Nucl.Acids Res. 17:477-508) can be prepared, for example, to increase therate of translation or to produce recombinant RNA transcripts havingdesirable properties, such as a longer half-life, as compared withtranscripts produced from a non-optimized sequence.

In another embodiment, the disclosure teaches a DNA construct comprisingtwo counterselectable markers, wherein the markers have beenindependently codon optimized for the microorganism and are sufficientlydistinct to prevent homologous recombination between the two markers. Insome embodiments, the markers have a maximum tandem identity length of500 base pairs when aligned with each other. In other embodiments, themarkers have a maximum tandem identity length of 250 base pairs whenaligned with each other. In other embodiments, the markers have amaximum tandem identity length of 100 base pairs when aligned with eachother. In other embodiments, the markers have a maximum tandem identitylength of 25 base pairs when aligned with each other. See for example,Koren, P. et al., (2000), Influence of homology size and polymorphism onplasmid integration in the yeast CYC1 DNA region. Current Genetics. 37.292-297.

Phenylalanyl-tRNA Synthetase

Phenylalanyl-tRNA synthetase is an enzyme that catalyzes theaminoacylation of tRNA^(Phe) with Phenylalanine. This “charging” or“loading” of the correct amino acid with its corresponding tRNA is animportant part of translation and the synthesis of proteins. Previouswork in E. coli has demonstrated that two mutations (A294G/T251A orA294G/T251S) to the sequence the α-subunit of phenylalanyl-tRNA ligase,PheS, can be used as a counterselectable marker due to the incorporationof 4-chloro-DL-phenylalanine (4CP) in place of phenylalanine duringtranslation, leading to cell death (Kast, Hennecke, 1991, Amino acidsubstrate specificity of Escherichia coli phenylalanyl-tRNA synthetasealtered by distinct mutations, J Mol Biol. 222(1):99-124, Miyazaki,2105, Molecular engineering of a PheS counterselection marker forimproved operating efficiency in Escherichia coli, Biotechniques,58(2):86-8). However, in Bacillus species those same PheS mutations arenot effective.

When PheS sequences were aligned and the corresponding mutations,A309G/T255S, were identified and introduced into B. subtilis pheS, andexpressed in B. amyloliquefaciens heterologously, this resulted inefficient killing in the presence of 4CP (Kharchenko M. S. et al., 2018.Improving the selection efficiency of the counter-selection marker pheS*for the genetic engineering of Bacillus amyloliquefaciens, J MicrobiolMethods. 148:18-21, and Zhou, C. et al., 2017, pheS*, an effectivehost-genotype-independent counter-selectable marker for marker-freechromosome deletion in Bacillus amyloliquefaciens. Appl MicrobiolBiotechnol.; 101(1):217-227).

In the absence of effective counterselectable marker, loop-out rates forBacillus range from 0.8% to 5%, making the reliable construction ofgenomic edits very challenging, especially in a high-throughput (HTP)context. Although some effective counterselection markers have beenreported in the literature for Bacillus species, many of those requirepre-existing genetic mutations such as upp, which requires the nativeupp be deleted from the genome for counterselection with 5-fluorouracil.Similarly, use of pyrF as a counterselection marker, requires that bothpyrF and pyrR be deleted in the strain of interest (Dong and Zhang 2014,Current development in genetic engineering strategies of Bacillusspecies, Microb Cell Fact. 13:63). However, expression of the mutantgene in B. subtilis resulted in false-positive clones due to homologousrecombination that replaced the mutant gene with the chromosomalwild-type pheS.

One embodiment of the disclosure provides for a HTP method forgenerating at least one scarless genomic edit in a Bacillus species.Methods for optimizing codons to improve expression in various hosts areknown in the art and described above (see also U.S. Pat. App. Pub. No.2007/0292918, incorporated herein by reference in its entirety). Thisallows for the introduction of stable genetic edits to the Bacillusgenome using either plasmid or linear DNA constructs for geneticengineering. The counterselection marker disclosed herein provides highefficiency genetic editing in Bacillus species, with PheS**CO leading tocell death solely in the presence of 4-chlorophenylalanine. While themethods disclosed herein have been successfully demonstrated to beeffective in B. licheniformis, and multiple strains of B. subtilis forthe construction of scarless genomic edits, one skilled in the art willrecognize they are applicable to all Bacillus species.

In an embodiment of the present disclosure, a plasmid or linear DNAconstruct is provided comprising a sequence of interest, a means forpositive selection, and a counterselectable marker, wherein thecounterselectable marker is an α-subunit of Phenylalanyl-tRNA ligase,(PheS) that has been codon optimized for Bacillus and further comprisesA309G/T255S mutations, and wherein the counterselectable marker isoperably linked to at least one promoter; transforming a Bacillusspecies with the DNA construct; selecting for a Bacillus strain havingintegrated the DNA construct based on the means for positive selection;growing the Bacillus strain having integrated the DNA construct in thepresence of 4-chlorphenylalanine to select for a Bacillus strain havingundergone a homologous recombination event excising the backbone of theplasmid containing the counterselectable marker to produce a loop-outstrain; and screening the loop-out strain for the presence of thesequence of interest to produce a modified Bacillus strain having atleast one scarless genomic edit.

For the codon optimization of PheS(**CO), the Bacillus amyloliquefaciensPheS sequence was used so the sequence is naturally codon optimized forBacillus species. For the codon optimization of PheS(**), SEQ ID NO: 3was input to the Integrated DNA Technologies (IDT) Codon OptimizationTool for optimization to B. subtilis.

In another embodiment, the PheS that has been codon optimized comprisesSEQ ID NO: 1 (PheS(**CO), or a sequence at least 90% identical thereto.In another embodiment, the PheS that has been codon optimized comprisesSEQ ID NO: 2 (PheS(**), or a sequence at least 75% identical thereto.Isolated nucleic acids and DNA constructs comprising these sequences arealso embodiments of the present disclosure.

In another embodiment, strains having integrated the DNA constructscomprising the PheS counterselectable marker are grown on or in mediacontaining between 1 mM and 20 mM 4-chlorphenyl alanine.

Bacillus Species

Bacillus is a genus of Gram-positive or Gram-variable spore-forming,aerobic or facultative anaerobic, rod-shaped bacteria. They areubiquitous in nature and have a wide range of physiologiccharacteristics and the ability to produce a variety of enzymes,antibiotics and metabolites, making them useful as a model organism andin many different industries, including for example, the medical,pharmaceutical, agricultural, and food industries. Non-limiting examplesof Bacillus that may be used with the methods disclosed herein includespecies of B. acidicele, B. acidicola, B. acidiproducens, B.acidocaldarius, B. acidoterrestris, B. aeolius, B. aerius, B.aerophilus, B. agaradhaerens, B. agri, B. aidingensis, B. akibai, B.alcalophilus, B. algicola, B. alginolyticus, B. alkalidiazotrophicus, B.alkalinitrilicus, B. alkalisediminis, B. alkalitelluris, B. altitudinis,B. alveayuensis, B. alvei, B. amyloliquefaciens, B. a. subsp.amyloliquefaciens, B. a. subsp. Plantarum, B. aminovorans, B.amylolyticus, B. andreesenii, B. aneurinilyticus, B. anthracis, B.aquimaris, B. arenosi, B. arseniciselenatis, B. arsenicus, B.aurantiacus, B. arvi, B. aryabhattai, B. asahii, B. atrophaeus, B.axarquiensis, B. azotofixans, B. azotoformans, B. badius, B. barbaricus,B. bataviensis, B. beijingensis, B. benzoevorans, B. beringensis, B.berkeleyi, B. beveridgei, B. bogoriensis, B. boroniphilus, B.borstelensis, B. brevis Migula, B. butanolivorans, B. canaveralius, B.carboniphilus, B. cecembensis, B. cellulosilyticus, B. centrosporus, B.cereus, B. chagannorensis, B. chitinolyticus, B. chondroitinus, B.choshinensis, B. chungangensis, B. cibi, B. circulans, B. clarkia, B.clausii, B. coagulans, B. coahuilensis, B. cohnii, B. composti, B.curdlanolyticus, B. cycloheptanicus, B. cytotoxicus, B. daliensis, B.decisifrondis, B. decolorationis, B. deserti, B. dipsosauri, B.drentensis, B. edaphicus, B. ehimensis, B. eiseniae, B. enclensis, B.endophyticus, B. endoradicis, B. farraginis, B. fastidiosus, B.fengqiuensis, B. firmus, B. flexus, B. foraminis, B. fordii, B.formosus, B. fortis, B. fumarioli, B. funiculus, B. fusiformis, B.galactophilus, B. galactosidilyticus, B. galliciensis, B. gelatini, B.gibsonii, B. ginseng, B. ginsengihumi, B. ginsengisoli, B.glucanolyticus, B. gordonae, B. gottheilii, B. graminis, B. halmapalus,B. haloalkaliphilus, B. halochares, B. halodenitrificans, B. halodurans,B. halophilus, B. halosaccharovorans, B. hemicellulosilyticus, B.hemicentroti, B. herbersteinensis, B. horikoshii, B. horneckiae, B.horti, B. huizhouensis, B. humi, B. hwajinpoensis, B. idriensis, B.indicus, B. infantis, B. infernus, B. insolitus, B. invictae, B.iranensis, B. isabeliae, B. isronensis, B. jeotgali, B. kaustophilus, B.kobensis, B. kochii, B. kokeshiiformis, B. koreensis, B. korlensis, B.kribbensis, B. krulwichiae, B. laevolacticus, B. larvae, B.laterosporus, B. lautus, B. lehensis, B. lentimorbus, B. lentus, B.licheniformis, B. ligniniphilus, B. litoralis, B. locisalis, B.luciferensis, B. luteolus, B. luteus, B. macauensis, B. macerans, B.macquariensis, B. macyae, B. malacitensis, B. mannanilyticus, B.marisflavi, B. marismortui, B. marmarensis, B. massiliensis, B.megaterium, B. mesonae, B. methanolicus, B. methylotrophicus, B.migulanus, B. mojavensis, B. mucilaginosus, B. muralis, B. murimartini,B. mycoides, B. naganoensis, B. nanhaiensis, B. nanhaiisediminis, B.nealsonii, B. neidei, B. neizhouensis, B. niabensis, B. niacin, B.novalis, B. oceanisediminis, B. odyssey, B. okhensis, B. okuhidensis, B.oleronius, B. oryzaecorticis, B. oshimensis, B. pabuli, B.pakistanensis, B. pallidus, B. pallidus, B. panacisoli, B. panaciterrae,B. pantothenticus, B. parabrevis, B. paraflexus, B. pasteurii, B.patagoniensis, B. peoriae, B. persepolensis, B. persicus, B. pervagus,B. plakortidis, B. pocheonensis, B. polygoni, B. polymyxa, B. popilliae,B. pseudalcalophilus, B. pseudofirmus, B. pseudomycoides, B.psychrodurans, B. psychrophilus, B. psychrosaccharolyticus, B.psychrotolerans, B. pulvifaciens, B. pumilus, B. purgationiresistens, B.pycnus, B. qingdaonensis, B. qingshengii, B. reuszeri, B. rhizosphaerae,B. rigui, B. ruris, B. safensis, B. salaries, B. salexigens, B.saliphilus, B. schlegelii, B. sediminis, B. selenatarsenatis, B.selenitireducens, B. seohaeanensis, B. shacheensis, B. shackletonii, B.siamensis, B. silvestris, B. simplex, B. spiralis, B. smithii, B. soli,B. solimangrovi, B. solisalsi, B. songklensis, B. sonorensis, B.sphaericus, B. sporothermodurans, B. stearothermophilus, B.stratosphericus, B. subterraneus, B. subtilis, B. s. subsp. Inaquosorum,B. s. subsp. Spizizenii, B. s. subsp. Subtilis, B. taeanensis, B.tequilensis, B. thermantarcticus, B. thermoaerophilus, B.thermoamylovorans, B. thermocatenulatus, B. thermocloacae, B.thermocopriae, B. thermodenitrificans, B. thermoglucosidasius, B.thermolactis, B. thermoleovorans, B. thermophilus, B. thermoruber, B.thermosphaericus, B. thiaminolyticus, B. thioparans, B. thuringiensis,B. tianshenii, B. trypoxylicola, B. tusciae, B. Validus, B.vallismortis, B. vedderi, B. velezensis, B. vietnamensis, B. vireti, B.vulcani, B. wakoensis, B. xiamenensis, B. xiaoxiensis, and B.zhanjiangensis.

Data suggests that Bacillus species may be sub-grouped based on 16S rDNAsequencing (Wei Wang M S. Phylogenetic relationships between Bacillusspecies and related genera inferred from 16s rDNA sequences. Braz JMicrobiol. 2009; 40(3):505-521). Thus, in another embodiment, themethods of scarless genomic editing disclosed herein may be used with aBacillus species selected from the group consisting of B. coagulans, B.ginsengihumi, B. shackletonii, B. aerius, B. aerophilus, B.stratosphericus, B. licheniformis, B. sonorensis, B. amyloliquefaciens,B. velezensis, B. atrophaeus, B. pumilus, B. safensis, B. altitudinis,B. vallismortis, B. subtilis, B. tequilensis, B. mojavensis, B.carbomphilus, B. oleronius, B. sporothermodurans, B. acidicola, B.aquimaris, B. vietnamensis, B. marisflavi, B. seohaeanensis, B.endophyticus, and B. humi.

Assembling & Cloning DNA Constructs

As will be understood by one skilled in the art, the methods ofgenerating scarless genomic edits disclosed herein may be used with anygenetic editing technology involving transformation of cells withexogenous DNA. In some embodiments, the present disclosure teachesmethods for constructing vectors capable of inserting a sequence ofinterest (e.g. containing a particular heterologous gene or a mutationto an endogenous gene) into the genome of host organisms. In someembodiments, the present disclosure teaches methods of cloning vectorscomprising the target DNA, homology arms, and at least one selectionmarker.

In some embodiments, the present disclosure is compatible with anyvector suited for transformation into the host organism. In certaininstances, the target DNA can be inserted into vectors, constructs orplasmids obtainable from any repository or catalogue product, such as acommercial vector (see e.g., DNA2.0 custom or GATEWAY® vectors).

In some embodiments, the methods of scarless genomic editing disclosedherein may employ an assembly/cloning method. Examples ofassembly/cloning methods include: i) type II conventional cloning, ii)type II S-mediated or “Golden Gate” cloning (see, e.g., Engler, C., R.Kandzia, and S. Marillonnet. 2008 “A one pot, one step, precisioncloning method with high-throughput capability”. PLos One 3:e3647;Kotera, I., and T. Nagai. 2008 “A high-throughput and single-tuberecombination of crude PCR products using a DNA polymerase inhibitor andtype IIS restriction enzyme.” J Biotechnol 137:1-7.; Weber, E., R.Gruetzner, S. Werner, C. Engler, and S. Marillonnet. 2011 Assembly ofDesigner TAL Effectors by Golden Gate Cloning. PloS One 6:e19722), iii)GATEWAY® recombination, iv) TOPO® cloning, exonuclease-mediated assembly(Aslanidis and de Jong 1990. “Ligation-independent cloning of PCRproducts (LIC-PCR).” Nucleic Acids Research, Vol. 18, No. 20 6069), v)homologous recombination, vi) non-homologous end joining, vii) Gibsonassembly (Gibson et al., 2009 “Enzymatic assembly of DNA molecules up toseveral hundred kilobases” Nature Methods 6, 343-345) or a combinationthereof. Modular type IIS based assembly strategies are disclosed in PCTPublication WO 2011/154147, the disclosure of which is incorporatedherein by reference.

Methods for Gene Editing

The disclosure provides methods for scarless gene editing, wherein asequence of interest is inserted into the host genome. As will beunderstood by one skilled in the art, the sequence of interest may be anendogenous gene that has been edited or it may be a heterologous gene.In some embodiments, the present disclosure teaches methods for geneediting by introducing, deleting, or replacing selected portions ofgenomic DNA. Gene editing (or mutations) may include single nucleotideinsertions or deletions, insertions or deletions of two or morenucleotides, insertion of a sequence encoding one or more proteins, adeletion of a sequence encoding one or more proteins, substitution of asingle nucleotide, or substitution of two or more nucleotides.

In other embodiments, the present disclosure teaches mutating selectedDNA regions outside of the host organism, and then inserting the mutatedsequence back into the host organism. For example, in some embodiments,the present disclosure teaches mutating native or synthetic promoters toproduce a range of promoter variants with various expression properties.In other embodiments, the present disclosure is compatible with singlegene optimization techniques, such as ProSAR (Fox et al. 2007.“Improving catalytic function by ProSAR-driven enzyme evolution.” NatureBiotechnology Vol 25 (3) 338-343, incorporated by reference herein).

In some embodiments, the selected regions of DNA are produced in vitrovia gene shuffling of natural variants, or shuffling with syntheticoligos, plasmid-plasmid recombination, virus plasmid recombination,virus-virus recombination. In other embodiments, the genomic regions areproduced via error-prone PCR.

In some embodiments, generating mutations in selected genetic regions isaccomplished by “reassembly PCR.” Briefly, oligonucleotide primers(oligos) are synthesized for PCR amplification of segments of a nucleicacid sequence of interest, such that the sequences of theoligonucleotides overlap the junctions of two segments. The overlapregion is typically about 10 to 100 nucleotides in length. Each of thesegments is amplified with a set of such primers. The PCR products arethen “reassembled” according to assembly protocols. In brief, in anassembly protocol, the PCR products are first purified away from theprimers, by, for example, gel electrophoresis or size exclusionchromatography. Purified products are mixed together and subjected toabout 1-10 cycles of denaturing, reannealing, and extension in thepresence of polymerase and deoxynucleoside triphosphates (dNTP's) andappropriate buffer salts in the absence of additional primers(“self-priming”). Subsequent PCR with primers flanking the gene are usedto amplify the yield of the fully reassembled and shuffled genes.

In some embodiments of the disclosure, mutated DNA regions, such asthose discussed above, are enriched for mutant sequences so that themultiple mutant spectrum, i.e. possible combinations of mutations, ismore efficiently sampled. In some embodiments, mutated sequences areidentified via a mutS protein affinity matrix (Wagner et al., NucleicAcids Res. 23(19):3944-3948 (1995); Su et al., Proc. Natl. Acad. Sci.(U.S.A.), 83:5057-5061(1986)) with a preferred step of amplifying theaffinity-purified material in vitro prior to an assembly reaction. Thisamplified material is then put into an assembly or reassembly PCRreaction as described in later portions of this application.

Thus, in some embodiments, “mutagenesis” or “genetic modification asused herein comprises all techniques known in the art for inducingmutations, including error-prone PCR mutagenesis,oligonucleotide-directed mutagenesis, site-directed mutagenesis, anditerative sequence recombination by any of the techniques describedherein.

Selectable Marker Gene

Selectable markers may be used with the methods of the presentdisclosure as a means for positive selection of transformants. Theselection marker may produce a RNA, peptide, or protein, or can providea binding site for RNA, peptides, proteins, inorganic and organiccompounds or compositions and the like. Examples of selectable markersinclude but are not limited to: (1) nucleic acid segments that encodeproducts which provide resistance against otherwise toxic compounds(e.g., antibiotics such as ampicillin, kanamycin, tetracycline,chloramphenicol, zeocin, streptomycin); (2) nucleic acid segments thatencode products which are otherwise lacking in the recipient cell (e.g.,tRNA genes, auxotrophic markers); (3) nucleic acid segments that encodeproducts which suppress the activity of a gene product; (4) nucleic acidsegments that encode products which can be readily identified (e.g.,phenotypic markers such as β-galactosidase, green fluorescent protein(GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP),and cell surface proteins); (5) nucleic acid segments that encodeproducts that bind other products which are otherwise detrimental tocell survival and/or function; (6) nucleic acid segments that encodenucleic acids that otherwise inhibit the activity of any of the nucleicacid segments resulting in a visible or selectable phenotype (e.g.,antisense oligonucleotides); (7) nucleic acid segments that encodeproducts that bind other products that modify a substrate (e.g.restriction endonucleases); (8) nucleic acid segments that can be usedto isolate or identify a desired molecule (e.g. specific protein bindingsites); (9) nucleic acid segments that encode a specific nucleotidesequence which can be otherwise non-functional (e.g., for PCRamplification of subpopulations of molecules); and (10) nucleic acidsegments, which when absent, directly or indirectly confer resistance orsensitivity to particular compounds.

In some embodiments, the selectable marker is an antibiotic resistancegene, for example, a chloramphenicol resistance gene, an ampicillinresistance gene, a tetracycline resistance gene, a zeocin resistancegene, a spectinomycin resistance gene a kanamycin resistance gene, atetracycline resistance gene, a neomycin resistance gene, a vancomycinresistance gene, a methicillin resistance gene, a penicillin resistancegene, an oxacillin resistance gene, erythromycin an erythromycinresistance gene, a linezolid resistance gene, a puromycin resistancegene, or a hygromycin resistance gene.

Non-limiting examples of selective agents include antibiotics, such asampicillin, tetracyclin, zeocin, spectinomycin, kanamycin, neomycin,vancomycin, methicillin, oxacillin, erythromycin, linezolid, puromycin,and hygromycin. Non-limiting examples of selectable marker genes includepyrG, hph, nat, amdS, nptII, niaD, and argB.

Promoters

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Insome embodiments, the promoter sequence consists of proximal and moredistal upstream elements, the latter elements often referred to asenhancers. Accordingly, an “enhancer” is a DNA sequence that canstimulate promoter activity, and may be an innate element of thepromoter or a heterologous element inserted to enhance the level ortissue specificity of a promoter. Promoters may be derived in theirentirety from a native gene, or be composed of different elementsderived from different promoters found in nature, or even comprisesynthetic DNA segments. It is understood by those skilled in the artthat different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity.

In some embodiments, the at least one promoter operably linked to thecounterselectable marker is constitutive, inducible, differentiallyinducible, endogenous, heterologous, synthetic, a dual promoter, or atandem promoter cluster.

In some embodiments, the methods of scarless genomic editing disclosedherein may be used with various promoters, including, for example,PliaG, P43, PliaI, PrpsF, Pspac, and Pspank (Vagner et al., 1998November A vector for systematic gene inactivation in Bacillus subtilis.Microbiology 144 (Pt 11), 3097-3104, Wu et al., 1991 Engineering aBacillus subtilis expression-secretion system with a strain deficient in6 extracellular proteases. J. Bacteriol. 173, 4952-4958). Otherpromoters include, for example, Plaps (Yang et al., 2013 Generation ofan artificial double promoter for protein expression in Bacillussubtilis through a promoter trap system. PLoS ONE 8:e56321), PBL₉ (Genget al., 2014 Mining tissue-specific contigs from peanut (Arachishypogaea L.) for promoter cloning by deep transcriptome sequencing.Plant Cell Physiol. 55, 1793-1801), Phag, PtufA, PcapD, PyqeY, PsodA,PfusA, PgapA, PahpF, PglnA, PamyE, and Pmdh (Meng F. et al., EnhancedExpression of Pullulanese in Bacillus subtilis by New Strong PromotersMined from Transcriptome Data, both Alone and in Combination,Microbiol., November 2018).

Where more than one counterselectable marker is used, embodiments hereininclude each counterselectable marker having a distinct operably linkedpromoter.

In some embodiments, dual promoters and/or promoter cassettes (tandempromoter clusters) may be used with the methods disclosed herein, suchas, for example, PhpaII-PamyQ (Zhang et al., 2017 High-levelextracellular protein production in Bacillus subtilis using an optimizeddual-promoter expression system. Microb. Cell Fact. 16:32), PgsiB-PHpaII(Guan C. R., et al, 2016 Construction of a highly active secretoryexpression system via an engineered dual promoter and a highly efficientsignal peptide in Bacillus subtilis. N. Biotechnol. 33, 372-379),PsodA+hag, PsodA+tugA, PsodA+fusA, PsodA+amyE, Phag+tufa, Phag+fusA,Phag+amyE, PtufA+fusA, PtufA+amyE, PfusA+amyE, PsodA+sodA, Phag+hag,PtufA+tufA, PfusA+fusA, PamyE+amyE, PsodA+hag+tufa, PsodA+hag+fusA,PsodA+hag+amyE, PsodA+tufA+fusA, PsodA+tufA+amyE, PsodA+fusA+amyE,Phag+tufA+fusA, Phag+tufA+amyE, Phag+fusA+amyE, PtufA+fusA+amyE,PsodA+sodA+sodA, Phag+hag+hag, PtufA+tufA+tufA, PamyE+amyE+amyE, andPfusA+fusA+fusA (Meng F. et al., Enhanced Expression of Pullulanese inBacillus subtilis by New Strong Promoters Mined from Transcriptome Data,both Alone and in Combination, Microbiol., November 2018).

In some embodiments, synthetic, reconstructed promoters may be used withthe methods disclosed herein (as in, for example, Liu D. et al., 2018Construction, Model-Based Analysis, and Characterization of a PromoterLibrary for Fine-Tuned Gene Expression in Bacillus subtilis, ACS Synth.Biol. 7, 7, 1785-1797) and others, for example Song Y., et al., 2016Promoter Screening from Bacillus subtilis in Various Conditions Huntingfor Synthetic Biology and Industrial Applications, PLoS ONE 11(7),Guiziou S., et al., 2016 A Part Toolbox to tune genetic expression inBacillus subtilis, Nucleic Acids Res., 44(15): 7495-7508.

Ribosomal Binding Sites

In some embodiments, the methods of scarless genomic editing disclosedherein utilize ribosomal binding sites.

Ribosomal binding sites (RBSs) are short sequences of nucleotides thatare located upstream of the start codon on an mRNA transcript that isresponsible for recruiting ribosomes and initiating translation ofprotein. Accordingly, they are important regulators of translation andprotein expression. However, RBSs can also interact with nearbynucleotides in the 5′UTR, the promoter or coding region of a gene toinfluence rates of transcription and/or translation. Through theseinteractions and resulting secondary structure, ribosomal binding sitescan “tune” expression of genes.

RBS libraries are common components of synthetic biology toolkits andhave been developed for various organisms. In addition, tools have beendeveloped for predicting synthetic RBSs that will interact favorablywith a sequence of interest (Salis et al., “Automated design ofsynthetic ribosome binding sites to control protein expression.” NatBiotechnol. 2009; 27:946-950. doi: 10.1038/nbt.1568).

Transcriptional Termination Sequences

In some embodiments, the methods of generating at least one scarlessgenomic edit disclosed herein utilize termination sequences.

In prokaryotes, two principal mechanisms, termed Rho-independent andRho-dependent termination, mediate transcriptional termination.Rho-independent termination signals do not require an extrinsictranscription-termination factor, as formation of a stem-loop structurein the RNA transcribed from these sequences along with a series ofUridine (U) residues promotes release of the RNA chain from thetranscription complex. Rho-dependent termination, on the other hand,requires a transcription-termination factor called Rho and cis-actingelements on the mRNA. The initial binding site for Rho, the Rhoutilization (rut) site, is an extended ('70 nucleotides, sometimes80-100 nucleotides) single-stranded region characterized by a highcytidine/low guanosine content and relatively little secondary structurein the RNA being synthesized, upstream of the actual terminatorsequence. When a polymerase pause site is encountered, terminationoccurs, and the transcript is released by Rho's helicase activity.

A transcriptional termination sequence may be any nucleotide sequence,which when placed transcriptionally downstream of a nucleotide sequenceencoding an open reading frame, causes the end of transcription of theopen reading frame. Such sequences are known in the art and may be ofprokaryotic, eukaryotic or phage origin. Examples of terminatorsequences include, but are not limited to, TgyrA (terminator sequence ofB. subtilis gyrA gene), TserO_aroC, TcodBA, arginine F gene (argF)terminator, PTH-terminator, pET-T7 terminator, T3-Tφ terminator,pBR322-P4 terminator, vesicular stomatitus virus terminator, rrnB-T1terminator, rrnC terminator, TTadc transcriptional terminator, andyeast-recognized termination sequences, such as Matα (α-factor)transcription terminator, native α-factor transcription terminationsequence, ADR1 transcription termination sequence, ADH2 transcriptiontermination sequence, and GAPD transcription termination sequence. Anon-exhaustive listing of transcriptional terminator sequences may befound in the iGEM registry, which is available at:partsregistry.org/Terminators/Catalog.

In some embodiments, transcriptional termination sequences may bepolymerase-specific or nonspecific, however, transcriptional terminatorsselected for use in the present embodiments should form a ‘functionalcombination’ with the selected promoter, meaning that the terminatorsequence should be capable of terminating transcription by the type ofRNA polymerase initiating at the promoter. The identity of thetranscriptional termination sequences used may also be selected based onthe efficiency with which transcription is terminated from a givenpromoter. For example, a heterologous transcriptional terminatorsequence may be provided transcriptionally downstream of the RNAencoding element to achieve a termination efficiency of at least 60%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% from a givenpromoter.

In some embodiments, the termination sequences are selected from TgyrA,Tsero_aroC, and TcodBA. Where more than one counterselectable marker isused, embodiments disclosed herein provide for each counterselectablemarker having a distinct termination sequence.

Transformation of Host Cells

Various methods for transformation are taught herein. In someembodiments, transformation of a competent cell involves heat-shock orelectroporation. In some embodiments, transformation is automated. Insome embodiments, competent cells are transformed using high-throughputelectroporation systems, for example, the VWR® High-throughputElectroporation Systems, BTX™, Bio-Rad® Gene Pulser MXcell™, or othermulti-well electroporation systems.

In some embodiments, the vectors of the present disclosure may beintroduced into the host cells using any of a variety of techniques,including transformation, transfection, transduction, viral infection,gene guns, or Ti-mediated gene transfer (see Christie, P. J., andGordon, J. E., 2014 “The Agrobacterium Ti Plasmids” Microbiol SPectr.2014; 2(6); 10.1128). Particular methods include calcium phosphatetransfection, DEAE-Dextran mediated transfection, lipofection, orelectroporation (Davis, L., Dibner, M., Battey, I., 1986 “Basic Methodsin Molecular Biology”). Other methods of transformation include forexample, lithium acetate transformation and electroporation See, e.g.,Gietz et al., Nucleic Acids Res. 27:69-74 (1992); Ito et al., J.Bacterol. 153:163-168 (1983); and Becker and Guarente, Methods inEnzymology 194:182-187 (1991). In some embodiments, transformed hostcells are referred to as recombinant host strains.

In some embodiments, the present disclosure teaches high-throughputtransformation of cells using the 96-well plate robotics platform andliquid handling machines of the present disclosure.

In some embodiments, the present disclosure teaches screeningtransformed cells with one or more selection markers as described above.In one such embodiment, cells transformed with a vector comprising achloramphenicol resistance marker [Chlor] are plated on media containingeffective amounts of the chloramphenicol antibiotic. In otherembodiments, cells are transformed with erythromycin and lincomycin[MLS] resistance markers and plated on media containing erythromycinand/or lincomycin. Colony forming units visible on antibiotic-lacedmedia are presumed to have incorporated the vector cassette into theirgenome. Insertion of the desired sequences can be confirmed via PCR,restriction enzyme analysis, and/or sequencing of the relevant insertionsite.

Looping Out of Selected Sequences (“Double Selection”)

In some embodiments, the present disclosure teaches methods ofcounterselection which favor strains having undergone a homologousrecombination excising the plasmid backbone to produce a “loop-out”strain. First, clones having integrated the DNA construct by a singlecrossover are selected for based on the means for positive selection,for example, antibiotic resistance. Clones are allowed to multiply toallow for a second crossover event, looping out the plasmid backbonecomprising the selective marker and counterselective marker. Whenselected clones are then grown on media corresponding to thecounterselective marker, for example 4-chloro-DL-phenylalanine, onlythose clones which have lost the counterselective marker (“loop-out”strains) will survive.

Additional looping out methods and techniques are known in the art, andare described in, for example, Tear et al. 2014 “Excision of UnstableArtificial Gene-Specific inverted Repeats Mediates Scar-Free GeneDeletions in Escherichia coli.” Appl. Biochem. Biotech. 175:1858-1867,and Nakashima et al. 2014 “Bacterial Cellular Engineering by GenomeEditing and Gene Silencing.” Int. J. Mol. Sci. 15(2), 2773-2793.

Screening Loop-Out Strains

In some embodiments, screening of the loop-out strains comprisessequencing, DNA fingerprinting, or phenotypic analysis.

In some embodiments, the present disclosure teaches whole-genomesequencing of the organisms described herein. In other embodiments, thepresent disclosure also teaches sequencing of plasmids, PCR products,and other oligos as quality controls to the methods of the presentdisclosure. Sequencing methods for large and small projects are wellknown to those in the art.

In some embodiments, any high-throughput technique for sequencingnucleic acids can be used in the methods of the disclosure. In someembodiments, the present disclosure teaches ultra deep sequencing of PCRamplicons to identify genetic variations. DNA sequencing techniquesinclude Next-Generation Sequencing (NGS) and classic dideoxy sequencingreactions (Sanger method) using labeled terminators or primers and gelseparation in slab or capillary; sequencing by synthesis usingreversibly terminated labeled nucleotides, pyrosequencing; 454sequencing; allele specific hybridization to a library of labeledoligonucleotide probes; sequencing by synthesis using allele specifichybridization to a library of labeled clones that is followed byligation; real time monitoring of the incorporation of labelednucleotides during a polymerization step; polony sequencing; and SOLiDsequencing.

In one aspect of the disclosure, high-throughput methods of sequencingare employed that comprise a step of spatially isolating individualmolecules on a solid surface where they are sequenced in parallel. Suchsolid surfaces may include nonporous surfaces (such as in Solexasequencing, e.g. Bentley et al, Nature, 456: 53-59 (2008) or CompleteGenomics sequencing, e.g. Drmanac et al, Science, 327: 78-81 (2010)),arrays of wells, which may include bead- or particle-bound templates(such as with 454, e.g. Margulies et al, Nature, 437: 376-380 (2005) orIon Torrent sequencing, U.S. patent publication 2010/0137143 or2010/0304982), micromachined membranes (such as with SMRT sequencing,e.g. Eid et al, Science, 323: 133-138 (2009)), or bead arrays (as withSOLiD sequencing or polony sequencing, e.g. Kim et al, Science, 316:1481-1414 (2007)).

In another embodiment, the methods of the present disclosure compriseamplifying the isolated molecules either before or after they arespatially isolated on a solid surface. Prior amplification may compriseemulsion-based amplification, such as emulsion PCR, or rolling circleamplification. Also taught is Solexa-based sequencing where individualtemplate molecules are spatially isolated on a solid surface, afterwhich they are amplified in parallel by bridge PCR to form separateclonal populations, or clusters, and then sequenced, as described inBentley et al (cited above) and in manufacturer's instructions (e.g.TruSeq™ Sample Preparation Kit and Data Sheet, Illumina, Inc., SanDiego, Calif., 2010); and further in the following references: U.S. Pat.Nos. 6,090,592; 6,300,070; 7,115,400; and EP0972081B1; which areincorporated by reference.

In another embodiment, screening of the loop-out strains comprisesphenotypic analysis. Phenotypic screening is a method used to identify astrain with a specific phenotypic trait and isolate it. Phenotype, asused herein, may apply to any cell property, including molecularphenotypes, such as the level of mRNA for a gene. Phenotypic analysismay comprise, for example, a step-wise process where individual loop-outstrains are cultured in media with specific substrates corresponding tothe phenotype of interest. Phenotypic assays have become more advancedand sophisticated and allow for high-throughput screening. For example,one skilled in the art may use a semi-automated bacterial phenotypicfingerprint (BPF) in conjunction with machine learning dataset analysis.

In another embodiment, the screening process may comprise amicroscopy-based high-throughput screening for alterations inmorphological phenotypes. See for example, Zahir, T., Camacho, R.,Vitale, R. et al. High-throughput time-resolved morphology screening inbacteria reveals phenotypic responses to antibiotics. Commun Biol 2, 269(2019).

In another embodiment, screening of the loop-out strains may comprise aDNA fingerprint analysis, also referred to as a microbial fingerprint orgenetic fingerprint. Methods of DNA fingerprint analysis are well knownin the art, and may comprise, for example, a restriction fragment lengthpolymorphism (RFLP), PCR, sequencing, probes, and/or blotting techniques(such as a Southern blot).

In another embodiment, the disclosure relates to microorganisms producedby the methods disclosed herein. In other embodiments, the disclosurerelates to Bacillus species produced by the methods disclosed herein. Insome embodiments, the microorganism or Bacillus species produced issubjected to further genetic modification. Such genetic modificationtechniques may comprise those described herein and/or other techniqueswell known in the art, including for example, direct gene editingmethods using natural or engineered nucleases (ZFNs, TALENS, or CRISPR).

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the disclosure and are not meant to limit the presentdisclosure in any fashion. Changes therein and other uses which areencompassed within the spirit of the disclosure, as defined by the scopeof the claims, will be recognized by those skilled in the art.

Example 1. PheS Confers Sensitivity to Counterselection Agent in B.subtilis

Replicating plasmids bearing PheS(**CO) driving by a range of promoterswere constructed using techniques well known in the art.

Plasmids were transformed into B. subtilis NRRL#BD-594 (530A) strain andtested for sensitivity. As shown in FIG. 1A-1D, a culture dilutionseries were spotted in six replicates onto LB media (FIG. 1A), LB mediawith the selective antibiotic chloramphenicol [Chlor] (FIG. 1B), LBmedia with the counterselection reagent 4CP (FIG. 1C), or thecombination of both chloramphenicol and 4CP (FIG. 1D), and incubated forone day. 530A strains with PheS constructs were unaffected on selectivemedia, but are significantly inhibited on 4CP (FIG. 1C). When bothchloramphenicol and 4CP were included in the media there was completecell death, as the plasmid could not be looped out due to the presenceof chloramphenicol (FIG. 1D). These results indicate that PheS(**CO) canserve as a counterselection marker in B. subtilis.

Example 2. PheS Confers Sensitivity to Counterselection Agent in B.subtilis Strain 168

Replicating plasmids bearing PheS(**CO) driving by a range of promotersand having different termination sequences were constructed as describedabove and transformed into B. subtilis 168 (B S168) strain and testedfor sensitivity. As shown in FIGS. 2A and 2B, a culture dilution serieswere spotted in six replicates onto LB media with either the selectiveantibiotic (Erythromycin and Lincomycin [MLS]) (FIG. 2A), or MLS+ thecounterselection reagent 4CP (FIG. 2B), and incubated for one day. BS168strains with PheS constructs were unaffected on selective media, butwere significantly inhibited on 4CP+MLS, where the strain is forced tomaintain the plasmid. These results indicate that PheS(**CO) can serveas a counterselection marker in B. subtilis in multiple geneticbackgrounds.

Example 3. PheS Confers Sensitivity to Counterselection Agent in B.licheniformis Strain DSM13

Replicating plasmids bearing PheS(**CO) driving by a range of promotersand having different termination sequences were constructed as describedabove and transformed into B. licheniformis DSM13 strain and tested forsensitivity. As shown in FIGS. 3A and 3B, culture dilution series werespotted in six replicates onto LB media with either the counterselectionreagent 4CP (FIG. 3A) or 4CP+ selective antibiotic MLS (FIG. 3B), andincubated for one day. All strains were unaffected on 4CP withoutpositive selection (FIG. 3A). The strains containing PheS(**CO) weresignificantly inhibited on 4CP with MLS selection in comparison to thecontrol strain (lacking counterselectable marker PheS), as the plasmidcannot be lost due to the presence of MLS (FIG. 3B). These resultsindicate that PheS(**CO) can serve as a counterselection marker in B.licheniformis.

Example 4. Loop-Out to Edit Rates with and without PheS Counterselectionin B. subtilis Strain S30A

To investigate loop-out to edit rates, B. subtilis strain S30 wastransformed with PheS(**CO) under the control of P43, PliaG, PrpsF, orPspac promoters and edit rates of two different loci, spoIIE and cotSwere analyzed. All constructs contained the terminator sequence of theB. subtilis DNA gyrase subunit A gene (TgyrA) to minimizetranscriptional read-through, and all constructs contained a positiveselection marker. Colonies were plated on LB plus Chloramphenicol toselect for positive transformants, which were subsequently grown onmedia comprising 10 mM 4-CP to select for loop-out strains. Colonieswere screened by NGS sequencing.

As shown below in Table 1, loop-out rates to correct edit for PheS(**CO)containing strains can reach upwards of (but not limited to) 89% ascompared to 2.5% for the markerless control.

TABLE 1 Loop-out to edit rates in B. subtilis S30A with PheS(**CO)counterselectable marker Number of Loop-out Colonies CounterselectableMarker to Edit Loci Tested Markerless 2.5%  spoIIE 570P43-PheS(**CO)-TgyrA 89% spoIIE 22 PliaG-PheS(**CO)-TgyrA 36% spoIIE 39PliaG-PheS(**CO)-TgyrA 46% cotS 34 PrpsF-PheS(**CO)-TgyrA 69% cotS 26Pspac-PheS(**CO)-TgyrA 30% cotS 48

Example 5. Loop-Out to Edit Rates with and without PheS Counterselectionin HTP Builds of B. subtilis Strain S30A

High throughput builds were attempted in B. subtilis strain S30A withconstructs that were identical besides the counterselection cassette;one did not express PheS (FIG. 4A) while the other comprised PheS(**CO)driven by the PliaG promoter (FIG. 4B). The integrated strains were thenserially diluted onto media having the counterselection reagent 4CP. Asshown in FIG. 4A, the markerless construct grows a lawn at alldilutions, indicating loop-out is not occurring. However, constructswith PheS(**CO) grow at a much lower density and successfully undergoloop-out. As indicated in Table 2 below, the overall build success ratefor the markerless edits was 0%, while the build success rate for theconstructs bearing PheS(**CO) was 86.3%.

TABLE 2 Loop-out rates for high throughput builds in B. subtilis strainS30A Build Success Number of Counterselectable Marker Rate StrainsAttempted Markerless   0% 86 PliaG-PheS(**CO)-TgyrA 86.3% 159

Example 6. Loop-Out Edit Rates with and without PheS Counterselection inB. licheniformis Strain DSM13

To investigate loop-out to edit rates in B. licheniformis, strain DSM13was transformed with PheS(**CO) under the control of P43 or PliaGpromoters and edit rates of ZBL30595 were analyzed. All constructscontained a terminator sequence of TserO_aroC or TcodBA to minimizetranscriptional read-through. As shown below in Table 3, loop-out ratesto correct edit for PheS(**CO) containing strains can reach upwards of(but not limited to) 11.9% as compared to 0.8% for the markerlesscontrol.

TABLE 3 Loop-out to edit rates in B. licheniformis DSM13 with PheS(**CO)counterselectable marker Number of Loop-out Colonies CounterselectableMarker to Edit Loci Tested Markerless 0.8% ZBL30595 378P43-PheS(**CO)-TserO_aroC 8.2% ZBL30595 196 P43-PheS(**CO)-TcodBA 9.8%ZBL30595 255 PliaG-PheS(**CO)-TcodBA 11.9% ZBL30595 126

Example 7. Expressing Two Copies of a Counterselectable Marker DecreasesBreakage in B. subtilis

In addition to PheS(**CO), a second codon optimized version of thecounterselectable marker, PheS(**) was generated as described herein,having an identical amino acid sequence. As shown in FIG. 5, analignment of SEQ ID NO: 1 (PheS(**CO)) and SEQ ID NO: 2 (PheS(**)) usingEMBL-EBI, (EMBOSS Water, Smith-Waterman algorithm) indicatesapproximately 75% shared identity, with a maximum tandem length of 14base pairs to prevent homologous recombination between the twosequences. However as will be understood by one skilled in the art,homologous recombination can be reduced or disrupted by even a singlebase pair difference (Koren, P. et al., (2000), Influence of homologysize and polymorphism on plasmid integration in the yeast CYC1 DNAregion. Current Genetics. 37. 292-297).

The 1×PheS(**CO) construct (FIG. 6A) was driven by PliaG promoter andhad a termination sequence of TgryA. The 2× construct comprisingPheS(**CO) and PheS(**) was driven by PliaG and PrpsF promoters,respectively, with TgyrA and TserOaroC termination sequences,respectively (FIG. 6B). Constructs were transformed into B. subtilisS30A and then spot plated with serial dilutions onto LB with 5 mM 4CP.For constructs with one copy of PheS(**CO) breakage occurred at an 8%rate ( 4/48), (FIG. 6A, denoted with black squares), however theconstruct comprising PheS(**CO) and PheS(**) breakage was lowered to 2%( 1/48), (FIG. 6B, denoted with a black square), demonstrating thatexpression two copies of a counterselectable marker in tandem havingdistinct nucleotide sequences decreases spontaneous breakage ofcounterselection, thereby reducing false positives and increasing theeffectiveness of counterselection.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

SEQ ID NO: 1 shows the nucleic acid sequence of the codon optimizedPheS(**CO) counterselection marker.

SEQ ID NO: 2 shows the nucleic acid sequence of the independently codonoptimized PheS(**) counterselection marker.

SEQ ID NO: 3 shows the corresponding amino acid sequence of SEQ ID NO: 1and SEQ ID NO: 2.

ADDITIONAL EMBODIMENTS OF THE DISCLOSURE

Other subject matter contemplated by the present disclosure is set outin the following numbered embodiments:

1. A high-throughput (HTP) method for generating at least one scarlessgenomic edit in a Bacillus species, comprising:

providing a plasmid or linear DNA construct comprising a sequence ofinterest, a means for positive selection, and a counterselectablemarker, wherein the counterselectable marker is an α-subunit ofPhenylalanyl-tRNA ligase, (PheS) that has been codon optimized forBacillus and further comprises A309G/T255S mutations, and wherein thecounterselectable marker is operably linked to at least one promoter;

transforming a Bacillus species with the DNA construct;

selecting for a Bacillus strain having integrated the DNA constructbased on the means for positive selection;

growing the Bacillus strain having integrated the DNA construct in thepresence of 4-chlorphenylalanine to select for a Bacillus strain havingundergone a homologous recombination event excising the backbone of theplasmid containing the counterselectable marker to produce a loop-outstrain; and screening the loop-out strain for the presence of thesequence of interest to produce a modified Bacillus strain having atleast one scarless genomic edit.

2. The method of embodiment 1, wherein the PheS that has been codonoptimized comprises SEQ ID NO: 1 (herein after referred to asPheS(**CO)), a sequence at least 90% identical thereto.

3. The method of embodiments 1 or 2, wherein the at least one promoteroperably linked to the counterselectable marker is constitutive,inducible, differentially inducible, endogenous, heterologous,synthetic, a dual promoter, or a tandem promoter cluster.

4. The method of any one of embodiments 1-3, wherein the at least onepromoter is selected from the group consisting of PliaG, P43, PliaI,PrpsF, Pspac, and Pspank.

5. The method of any one of embodiments 1-4, wherein the at least onepromoter is a dual promoter or tandem promoter selected from the groupconsisting of PHpaII-PamyQ′ and PgsiB-PHpaII.

6. The method of any one of embodiments 1-5, wherein the DNA constructfurther comprises a ribosomal binding site.

7. The method of any one of embodiments 1-6, wherein the DNA constructfurther comprises a termination sequence.

8. The method of embodiment 7, wherein the termination sequence isselected from the group consisting of TgyrA, Tsero_aroC, and TcodBA.

9. The method of any one of embodiments 1-8, wherein the Bacillusspecies is transformed with the construct using natural competence,conjugation, electroporation, transduction, or protoplasttransformation.

10. The method of any one of embodiments 1-9, wherein the strain havingintegrated the DNA construct is grown on or in media containing between1 mM and 20 mM 4-chlorphenylalanine.

11. The method of any one of embodiments 1-10, wherein screening of theloop-out strain comprises sequencing, DNA fingerprinting, or phenotypicanalysis.

12. The method of any one of embodiments 1-11, wherein the sequence ofinterest is an endogenous gene having a least one mutation sequence.

13. The method of embodiment 12, wherein the mutation sequence comprisesa mutation selected from the group consisting of:

a. a single nucleotide insertion;

b. an insertion of two or more nucleotides;

c. an insertion of a nucleic acid sequence encoding one or moreproteins;

d. a single nucleotide deletion;

e. a deletion of two or more nucleotides;

f. a deletion of one or more coding sequences;

g. a substitution of a single nucleotide;

h. a substitution of two or more nucleotides;

i. two or more non-contiguous insertions, deletions, and/orsubstitutions; and

j. any combination thereof.

14. The method of any one of embodiments 1-13, wherein the sequence ofinterest is a heterologous gene.

15. A genetically modified Bacillus strain produced by any one of themethods of embodiments 1-14.

16. The genetically modified Bacillus strain of embodiment 15, whereinthe Bacillus species is selected from the group consisting of B.coagulans, B. ginsengihumi, B. shackletonii, B. aerius, B. aerophilus,B. stratosphericus, B. licheniformis, B. sonorensis, B.amyloliquefaciens, B. velezensis, B. atrophaeus, B. pumilus, B.safensis, B. altitudinis, B. vallismortis, B. subtilis, B. tequilensis,B. mojavensis, B. carboniphilus, B. oleronius, B. sporothermodurans, B.acidicola, B. aquimaris, B. vietnamensis, B. marisflavi, B.seohaeanensis, B. endophyticus, and B. humi.17. The modified Bacillus strain of embodiment 16, wherein the modifiedBacillus strain is subjected to further genetic modification.18. A DNA construct comprising the counterselection marker PheS(**CO)comprising SEQ ID NO: 1, or a sequence at least 90% identical thereto.19. A Bacillus strain having the DNA construct of embodiment 18.20. The method of embodiment 1, wherein the PheS that has been codonoptimized comprises SEQ ID NO: 2 (herein after referred to as PheS(**)),or a sequence at least 75% identical thereto.21. The method of embodiment 20, wherein the at least one promoteroperably linked to the counterselectable marker is constitutive,inducible, differentially inducible, endogenous, heterologous,synthetic, a dual promoter, or a tandem promoter cluster.22. The method of any one of embodiments 20-21, wherein the at least onepromoter is selected from the group consisting of PliaG, P43, PliaI,PrpsF, Pspac, and Pspank.23. The method of any one of embodiments 20-22, wherein the promoter isa dual promoter or tandem promoter selected from the group consisting ofPHpaII-PamyQ′ and PgsiB-PHpaII.24. The method of any one of embodiments 20-23, wherein the DNAconstruct further comprises a ribosomal binding site.25. The method of any one of embodiments 20-24, wherein the DNAconstruct further comprises a termination sequence.26. The method of embodiment 25, wherein the termination sequence isselected from the group consisting of TgyrA, Tsero_aroC, and TcodBA.27. The method of any one of embodiments 20-26, wherein the Bacillusspecies is transformed with the construct using natural competence,conjugation, electroporation, transduction, or protoplasttransformation.28. The method of any one of embodiments 20-27, wherein the strainhaving integrated the DNA construct is grown on or in media containingbetween 1 mM and 20 mM 4-chlorphenylalanine.29. The method of any one of embodiments 20-28, wherein the screening ofthe loop-out strain comprises sequencing, DNA fingerprinting, orphenotypic analysis.30. The method of any one of embodiments 20-29, wherein the sequence ofinterest is an endogenous gene having a least one mutation sequence.31. The method of embodiment 30, wherein the mutation sequence comprisesa mutation selected from the group consisting of:

a. a single nucleotide insertion;

b. an insertion of two or more nucleotides;

c. an insertion of a nucleic acid sequence encoding one or moreproteins;

d. a single nucleotide deletion;

e. a deletion of two or more nucleotides;

f. a deletion of one or more coding sequences;

g. a substitution of a single nucleotide;

h. a substitution of two or more nucleotides;

i. two or more non-contiguous insertions, deletions, and/orsubstitutions; and

j. any combination thereof.

32. The method of any one of embodiments 20-29, wherein the sequence ofinterest is a heterologous gene.

33. A genetically modified Bacillus strain produced by any one of themethods of embodiments 20-33.

34. The genetically modified Bacillus strain of embodiment 33, whereinthe Bacillus species is selected from the group consisting of B.coagulans, B. ginsengihumi, B. shackletonii, B. aerius, B. aerophilus,B. stratosphericus, B. licheniformis, B. sonorensis, B.amyloliquefaciens, B. velezensis, B. atrophaeus, B. pumilus, B.safensis, B. altitudinis, B. vallismortis, B. subtilis, B. tequilensis,B. mojavensis, B. carboniphilus, B. oleronius, B. sporothermodurans, B.acidicola, B. aquimaris, B. vietnamensis, B. marisflavi, B.seohaeanensis, B. endophyticus, and B. humi.35. The modified Bacillus strain of embodiment 34, wherein the modifiedBacillus strain is subjected to further genetic modification.36. A DNA construct comprising the counterselection marker PheS(**)comprising SEQ ID NO: 2, or a sequence at least 75% identical thereto.37. A Bacillus strain having the DNA construct of embodiment 36.38. A method for generating at least one scarless genomic edit in aBacillus species, comprising:

providing a plasmid or linear DNA construct comprising a sequence ofinterest, a means for positive selection, and two counterselectablemarkers, wherein the counterselectable markers are an α-subunit ofphenylalanyl-tRNA ligase, (PheS) that have been codon optimized forBacillus and further comprise A309G/T255S mutations, wherein thecounterselectable markers have a maximum tandem identity length of 500base pairs when aligned with each other, and wherein eachcounterselectable marker is operably linked to at least one promoter;

transforming a Bacillus species with the DNA construct;

selecting for a Bacillus strain having integrated the DNA constructbased on the means for positive selection;

growing the Bacillus strain having integrated the DNA construct in thepresence of 4-chlorphenylalanine to select for a Bacillus strain havingundergone a homologous recombination event excising the backbone of theplasmid containing the counterselectable marker to produce a loop-outstrain; and

screening the loop-out strain for the presence of the sequence ofinterest to produce a modified Bacillus strain having at least onescarless genomic edit.

39. The method of embodiment 38, wherein one counterselectable markercomprises SEQ ID NO: 1, or a sequence at least 90% identical thereto,and the other comprises SEQ ID NO: 2, or a sequence at least 75%identical thereto.

40. The method of embodiments 38 or 39, wherein the at least onepromoter operably linked to each counterselectable marker isconstitutive, inducible, differentially inducible, endogenous,heterologous, synthetic, a dual promoter, or a tandem promoter cluster.41. The method of any one of embodiments 38-40, wherein the at least onepromoter is selected from the group consisting of PliaG, P43, PliaI,PrpsF, Pspac, and Pspank.42. The method of any one of embodiments 38-41, wherein the at least onepromoter is a dual promoter or tandem promoter selected from the groupconsisting of PHpaII-PamyQ′ and PgsiB-PHpaII.43. The method of any one of embodiments 38-42, wherein eachcounterselectable marker has a distinct operably linked promoter.44. The method of any one of embodiments 38-43, wherein the DNAconstruct further comprises a ribosomal binding site.45. The method of any one of embodiments 38-44, wherein each of thecounterselectable markers further comprises a termination sequence.46. The method of embodiment 45, wherein the termination sequence isselected from the group consisting of TgyrA, Tsero_aroC, and TcodBA.47. The method of embodiment 45 or 46, wherein each counterselectablemarker has a distinct termination sequence.48. The method of any one of embodiments 38-47, wherein the Bacillusspecies is transformed with the construct using natural competence,conjugation, electroporation, transduction, or protoplasttransformation.49. The method of any one of embodiments 38-48, wherein the strainhaving integrated the DNA construct is grown on or in media containingbetween 1 mM and 20 mM 4-chlorphenylalanine.50. The method of any one of embodiments 38-49, wherein screening of theloop-out strain comprises sequencing, DNA fingerprinting, or phenotypicanalysis.51. The method of any one of embodiments 38-50, wherein the sequence ofinterest is an endogenous gene having a least one mutation sequence.52. The method of embodiment 51, wherein the mutation sequence comprisesa mutation selected from the group consisting of:

a. a single nucleotide insertion;

b. an insertion of two or more nucleotides;

c. an insertion of a nucleic acid sequence encoding one or moreproteins;

d. a single nucleotide deletion;

e. a deletion of two or more nucleotides;

f. a deletion of one or more coding sequences;

g. a substitution of a single nucleotide;

h. a substitution of two or more nucleotides;

i. two or more non-contiguous insertions, deletions, and/orsubstitutions; and

j. any combination thereof.

53. The method of any one of embodiments 38-52, wherein the sequence ofinterest is a heterologous gene.

54. A genetically modified Bacillus strain produced by any one of themethods of embodiments 38-53.

55. The genetically modified Bacillus strain of embodiment 54, whereinthe Bacillus species is selected from the group consisting of B.coagulans, B. ginsengihumi, B. shackletonii, B. aerius, B. aerophilus,B. stratosphericus, B. licheniformis, B. sonorensis, B.amyloliquefaciens, B. velezensis, B. atrophaeus, B. pumilus, B.safensis, B. altitudinis, B. vallismortis, B. subtilis, B. tequilensis,B. mojavensis, B. carboniphilus, B. oleronius, B. sporothermodurans, B.acidicola, B. aquimaris, B. vietnamensis, B. marisflavi, B.seohaeanensis, B. endophyticus, and B. humi.56. The modified Bacillus strain of embodiment 55, wherein the modifiedBacillus strain is subjected to further genetic modification.57. A DNA construct comprising the counterselection marker PheS(**CO)comprising SEQ ID NO: 1, or a sequence at least 90% identical thereto,and the counterselection marker PheS(**) comprising SEQ ID NO: 2, or asequence at least 75% identical thereto.58. A Bacillus strain having the DNA construct of embodiment 57.59. A high-throughput (HTP) method for generating at least one scarlessgenomic edit in a microorganism, comprising:

providing a plasmid or linear DNA construct comprising a sequence ofinterest and at least one counterselectable marker, wherein the at leastone counterselectable marker is a homolog of the α-subunit ofPhenylalanyl-tRNA ligase, (PheS) that has been codon optimized for amicroorganism, and further comprises homologous mutations correspondingto A309G/T255S of Bacillus PheS, and wherein the at least onecounterselectable marker is operably linked to at least one promoter;

transforming the microorganism with the DNA construct to produce atransformed strain;

growing the transformed strain in the presence of 4-chlorphenylalanineto select for a strain having undergone a recombination event excisingthe backbone of the plasmid containing the at least onecounterselectable marker to produce a loop-out strain; and

screening the loop-out strain for the presence of the sequence ofinterest to produce a scarless genetically modified microorganism.

60. The method of embodiment 59, wherein the DNA construct comprises twocounterselectable markers, wherein the markers have been independentlycodon optimized for the microorganism and are sufficiently distinct toprevent homologous recombination between the two markers.61. The method of embodiment 60, wherein the two counterselectablemarkers have a maximum tandem identity length of 500 base pairs whenaligned with each other.62. The method of embodiment 60, wherein the two counterselectablemarkers have a maximum tandem identity length of 250 base pairs whenaligned with each other.63. The method of embodiment 60, wherein the two counterselectablemarkers have a maximum tandem identity length of 100 base pairs whenaligned with each other.64. The method of embodiment 60, wherein the two counterselectablemarkers have a maximum tandem identity length of 25 base pairs whenaligned with each other.65. The method of any one of embodiments 59-64, wherein the DNAconstruct further comprises a means for positive selection.66. The method of any one of embodiments 59-65, wherein the DNAconstruct further comprises a ribosomal binding site.67. The method of any one of embodiments 59-66, wherein the at least onecounterselectable marker further comprises a termination sequence.68. The method of any one of embodiments 59-67, wherein the sequence ofinterest is an endogenous gene having a least one mutation sequence.69. The method of embodiment 68, wherein the mutation sequence comprisesa mutation selected from the group consisting of:

a. a single nucleotide insertion;

b. an insertion of two or more nucleotides;

c. an insertion of a nucleic acid sequence encoding one or moreproteins;

d. a single nucleotide deletion;

e. a deletion of two or more nucleotides;

f. a deletion of one or more coding sequences;

g. a substitution of a single nucleotide;

h. a substitution of two or more nucleotides;

i. two or more non-contiguous insertions, deletions, and/orsubstitutions; and

j. any combination thereof.

70. The method of any one of embodiments 59-67, wherein the sequence ofinterest is a heterologous gene.

71. A microorganism produced by the method of any one of embodiments59-70.

72. The method of embodiment 71, wherein the microorganism is a Bacillusspecies.

73. The method of embodiment 72, wherein the sequence of interest is aDNA fragment having homology to a genomic locus of the Bacillus species.

74. The method of any one of embodiments 59-73, wherein the screeningthe loop-out strain comprises sequencing, DNA fingerprinting, orphenotypic analysis.

75. The method of any one of embodiments 59-74, wherein the methodresults in greater than 2.5% of microorganisms containing a scarlessgenetic modification.

76. A high-throughput (HTP) method for generating at least one scarlessgenomic edit in a microorganism, comprising:

providing a plasmid or linear DNA construct comprising a sequence ofinterest, a means for positive selection, and two counterselectablemarkers, wherein each of the counterselectable markers have beenindependently codon optimized for the microorganism and have a maximumtandem identity length of 500 base pairs when aligned with each other,and wherein each counterselectable marker is operably linked to at leastone promoter;

transforming the microorganism with the DNA construct;

selecting for a microorganism strain having integrated the DNA constructbased on the means for positive selection;

selecting for a microorganism having undergone a homologousrecombination event excising the backbone of the plasmid containing thecounterselectable markers to produce a loop-out strain; and

screening the loop-out strain for the presence of the sequence ofinterest to produce a modified microorganism having at least onescarless genomic edit.

77. The method of embodiment 76, wherein the microorganism is a Bacillusspecies.

78. The method of embodiment 77, wherein the counterselectable markersare an α-subunit of phenylalanyl-tRNA ligase, (PheS) and furthercomprise A309G/T255S mutations.

79. The method of embodiment 78, wherein one counterselectable markercomprises SEQ ID NO: 1, or a sequence at least 90% identical thereto,and the other comprises SEQ ID NO: 2, or a sequence at least 75%identical thereto.

80. The method of any one of embodiments 76-79, wherein the at least onepromoter operably linked to each counterselectable marker isconstitutive, inducible, differentially inducible, endogenous,heterologous, synthetic, a dual promoter, or a tandem promoter cluster.81. The method of any one of embodiments 76-80, wherein the at least onepromoter is selected from the group consisting of PliaG, P43, PliaI,PrpsF, Pspac, and Pspank.82. The method of any one of embodiments 76-81, wherein eachcounterselectable marker has a distinct operably linked promoter.83. The method of any one of embodiments 76-82, wherein each of thecounterselectable markers further comprises a termination sequence.84. The method of embodiment 83, wherein the termination sequence isselected from the group consisting of TgyrA, Tsero_aroC, and TcodBA.85. The method of embodiment 83 or 84, wherein each counterselectablemarker has a distinct termination sequence.86. The method of any one of embodiments 78-85, wherein the strainhaving integrated the DNA construct is grown on or in media containingbetween 1 mM and 20 mM 4-chlorphenylalanine.87. The method of any one of embodiments 76-86, wherein screening of theloop-out strain comprises sequencing, DNA fingerprinting, or phenotypicanalysis.88. The method of any one of embodiments 76-87, wherein the sequence ofinterest is an endogenous gene or a heterologous gene, wherein theendogenous gene has at least one mutation sequence selected from thegroup consisting of:

a. a single nucleotide insertion;

b. an insertion of two or more nucleotides;

c. an insertion of a nucleic acid sequence encoding one or moreproteins;

d. a single nucleotide deletion;

e. a deletion of two or more nucleotides;

f. a deletion of one or more coding sequences;

g. a substitution of a single nucleotide;

h. a substitution of two or more nucleotides;

i. two or more non-contiguous insertions, deletions, and/orsubstitutions; and

j. any combination thereof.

89. A genetically modified Bacillus strain produced by the method of anyone of embodiments 77-88.

90. The modified Bacillus strain of claim 89, wherein the modifiedBacillus strain is subjected to further genetic modification.

91. A genetically modified microorganism produced by the method of anyone of embodiments 76-90.

92. An isolated nucleic acid comprising SEQ ID NO: 1, or a sequence atleast 90% identical thereto.

93. An isolated nucleic acid comprising SEQ ID NO: 2, or a sequence atleast 75% identical thereto.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes. However, mention of any reference,article, publication, patent, patent publication, and patent applicationcited herein is not, and should not be taken as, an acknowledgment orany form of suggestion that they constitute valid prior art or form partof the common general knowledge in any country in the world. Further,PCT/US2016/065465 (WO 2017/100377 A1), filed Dec. 7, 2016, and entitled:Microbial Strain Improvement By A HTP Genomic Engineering Platform ishereby incorporated by reference.

What is claimed is:
 1. A high-throughput (HTP) method for generating atleast one scarless genomic edit in a Bacillus species, comprising:providing a plasmid or linear DNA construct comprising a sequence ofinterest, a positive selectable marker, and two copies of the α-subunitof phenylalanyl-tRNA ligase (PheS) as counterselectable markers, whereineach of the counterselectable markers have been independently codonoptimized for the Bacillus species and have a maximum continuousidentity length of 500 base pairs when aligned with each other, andwherein each counterselectable marker is operably linked to at least onepromoter; transforming the Bacillus species with the DNA construct;selecting for a Bacillus strain having integrated the DNA constructbased on the positive selectable marker; selecting for a Bacillus strainhaving undergone a homologous recombination event excising the backboneof the plasmid containing the counterselectable markers to produce aloop-out strain; and screening the loop-out strain for the presence ofthe sequence of interest to produce a modified Bacillus strain having atleast one scarless genomic edit.
 2. The method of claim 1, wherein theBacillus species is selected from the group consisting of B. coagulans,B. ginsengihumi, B. shackletonii, B. aerius, B. aerophilus, B.stratosphericus, B. licheniformis, B. sonorensis, B. amyloliquefaciens,B. velezensis, B. atrophaeus, B. pumilus, B. safensis, B. altitudinis,B. vallismortis, B. subtilis, B. tequilensis, B. mojavensis, B.carboniphilus, B. oleronius, B. sporothermodurans, B. acidicola, B.aquimaris, B. vietnamensis, B. marisflavi, B. seohaeanensis, B.endophyticus, and B. humi.
 3. The method of claim 1, wherein thecounterselectable markers further comprise A309G/T255S mutations.
 4. Themethod of claim 3, wherein one counterselectable marker comprises SEQ IDNO: 1, or a sequence at least 90% identical thereto, and the othercomprises SEQ ID NO: 2, or a sequence at least 75% identical thereto. 5.The method of claim 1, wherein the at least one promoter operably linkedto each counterselectable marker is constitutive, inducible,differentially inducible, endogenous, heterologous, synthetic, a dualpromoter, or a tandem promoter cluster.
 6. The method of claim 1,wherein the at least one promoter is selected from the group consistingof PliaG, P43, PliaI, PrpsF, Pspac, and Pspank.
 7. The method of claim1, wherein each counterselectable marker has a distinct operably linkedpromoter.
 8. The method of claim 1, wherein each of thecounterselectable markers further comprises a termination sequence. 9.The method of claim 8, wherein the termination sequence is selected fromthe group consisting of TgyrA, Tsero_aroC, and TcodBA.
 10. The method ofclaim 8, wherein each counterselectable marker has a distincttermination sequence.
 11. The method of claim 3, wherein the strainhaving integrated the DNA construct is grown on or in media containingbetween 1 mM and 20 mM 4-4-chloropheylalanine.
 12. The method of claim1, wherein screening of the loop-out strain comprises sequencing, DNAfingerprinting, or phenotypic analysis.
 13. The method of claim 1,wherein the sequence of interest is an endogenous gene or a heterologousgene, wherein the endogenous gene has at least one mutation sequenceselected from the group consisting of: a. a single nucleotide insertion;b. an insertion of two or more nucleotides; c. an insertion of a nucleicacid sequence encoding one or more proteins; d. a single nucleotidedeletion; e. a deletion of two or more nucleotides; f. a deletion of oneor more coding sequences; g. a substitution of a single nucleotide; h. asubstitution of two or more nucleotides; i. two or more non-contiguousinsertions, deletions, and/or substitutions; and j. any combinationthereof.
 14. The method of claim 1, wherein the two copies of theα-subunit of phenylalanyl-tRNA ligase (PheS) are consecutive.
 15. Themethod of claim 1, wherein the amino acid sequences of the two copies ofthe α-subunit of phenylalanyl-tRNA ligase (PheS) are identical.